1st Edition

Biomolecular Kinetics A Step-by-Step Guide

By Clive R. Bagshaw Copyright 2017
    470 Pages
    by CRC Press

    470 Pages
    by CRC Press

    "a gem of a textbook which manages to produce a genuinely fresh, concise yet comprehensive guide"
    –Mark Leake, University of York

    "destined to become a standard reference…. Not just a ‘how to’ handbook but also an accessible primer in the essentials of kinetic theory and practice."
    –Michael Geeves, University of Kent

    "covers the entire spectrum of approaches, from the traditional steady state methods to a thorough account of transient kinetics and rapid reaction techniques, and then on to the new single molecule techniques"
    –Stephen Halford, University of Bristol

    This illustrated treatment explains the methods used for measuring how much a reaction gets speeded up, as well as the framework for solving problems such as ligand binding and macromolecular folding, using the step-by-step approach of numerical integration. It is a thoroughly modern text, reflecting the recent ability to observe reactions at the single-molecule level, as well as advances in microfluidics which have given rise to femtoscale studies. Kinetics is more important now than ever, and this book is a vibrant and approachable entry for anyone who wants to understand mechanism using transient or single molecule kinetics without getting bogged down in advanced mathematics.

    Clive R. Bagshaw is Emeritus Professor at the University of Leicester, U.K., and Research Associate at the University of California at Santa Cruz, U.S.A.


    What can we learn from kinetics?

    Kinetic measurements: the transient- and the steady-state.

    Kinetic measurements: ensemble and single-molecule approaches.


    One-step irreversible reaction

    One-step reversible reaction

    Irreversible binding reaction

    Reversible binding reaction

    Two-step irreversible reaction

    Two-step reversible reaction

    Two-step reversible binding reaction: induced fit

    Two-step reversible binding reaction: conformational selection

    Thermodynamic boxes

    Alternative binding mode

    Competitive binding

    Displacement reactions

    Multi-site ligand binding

    Multi-step reversible reactions


    Basic thermodynamics

    The significance of Free Energy, D G

    Thermal energy

    Reaction kinetics and the transition state

    Time scales of binding kinetics: diffusion-controlled reactions

    The Time Scales of Macromolecular Conformational Changes

    Force dependence of rate constants

    The basis of enzyme catalysis


    Enzyme catalysis with a single substrate

    Enzyme catalysis with two substrates

    Identification of the rate-limiting step

    The use of isotopes

    Enzyme activators and inhibitors

    Allosteric and Cooperative reactions


    Protein folding

    Polymerization reactions

    Nucleic acid dynamics

    Motor proteins

    Membrane Transporters, pumps and channels

    Biological Clocks

    Kinetics in vivo


    Sample preparation

    Preliminary characterization

    Characterization of equilibrium binding constants

    Steady-state enzyme kinetics

    Transient kinetic assays

    Tuning the system for measurement

    The use of indicators

    Single-molecule assays


    The basic components

    Mixing techniques

    Perturbation techniques

    Flash photolysis

    Components for optical detection

    Detection methods


    Basic statistical concepts

    Analysis of linear functions

    Least-squares fitting to analytical expressions

    Global fitting to analytical equations

    Fitting data directly to kinetic models

    Multi-wavelength analysis



    Instrumentation Single-molecule force measurements

    Analysis of single-molecule kinetic data

    Analysis of complex time trajectories

    Autocorrelation analysis

    Single-molecule enzymology


    Biochemical time scales

    DNA binding proteins

    Induced Fit versus Conformational Selection revisited


    Some useful physical constants

    Kinetic instrument suppliers

    Kinetic software

    Use of spreadsheets in kinetic analysis


    Clive Bagshaw obtained a BSc in Biochemistry at the University of Birmingham and a PhD from the University of Bristol. His thesis involved investigation of muscle myosin ATPase activity using transient kinetic methods. The resultant kinetic mechanism, proposed together with his supervisor David Trentham, became textbook information. Following post-doctoral periods at the Universities of Pennsylvania and Oxford, he obtained a lectureship at the University of Leicester, where he taught courses on protein structure and function for 30 years. He continued his research of myosin ATPase activity, initially using a home-built stopped-flow apparatus based on the Gutfreund design. Subsequently, his instrument arsenal was extended, in collaboration with other faculty members, to include commercial stopped-flow, quenched-flow, temperature-jump and flash photolysis instruments. His own research was extended into the mechanisms of myosin ATPase regulation by Ca2+ ions and the kinetics of non-muscle myosins. He wrote a short textbook on Muscle Contraction in 1982, which was revised and extended in 1993 to include in vitro motility assays. The latter was inspired by a sabbatical period in Jim Spudich's laboratory at Stanford University. On return to Leicester, he constructed a total internal reflectance fluorescence microscope to study ATP turnover by myosin at the single-molecule level. In the search for suitable fluorescence probes, he also studied the photophysics of a number of variants of green fluorescent proteins. More recently, he has been involved in collaborative research on other cytoskeletal proteins, S100 proteins, splicing factors and DNA-based photonic wires. From year 2000, Clive has been an instructor on the biannual EMBO Practical Course on Transient Kinetics held at the University of Kent. In 2002 he obtained a personal chair in physical biochemistry and retired from Leicester University in 2011. This transition was catalyzed by a sabbatical at the University of California at Santa Cruz in the laboratory of Michael Stone, which led to an honorary research position that provided the academic resources and freedom to write this book on kinetics. He continues to assist in research using single-molecule methods to study telomerases and enjoy the spectacular natural history of the Monterey Bay.

    "a unique and innovative contribution to the field... the first to my knowledge that covers the entire spectrum of approaches, from the traditional steady state methods to a thorough account of transient kinetics and rapid reaction techniques, and then on to the new single molecule techniques… a significant contribution."
    —Prof. Stephen Halford, University of Bristol

    " Enzyme kinetics is regaining its rightful place at the center of molecular and cellular biology…. Thus, a new generation of students will benefit from Clive Bagshaw's Biomolecular Kinetics: A Step-By-Step Guide"
    —Jonathon Howard, Eugene Higgins Professor of Molecular Biophysics & Biochemistry, Yale

    "Comprehensive and well written."
    —Enrico Di Cera, MD, Alice A. Doisy Professor and Chairman, Department of Biochemistry and Molecular Biology, Saint Louis University

    "This book is destined to become a standard reference on the bookshelf of any laboratory with an interest in kinetic approaches to biology. Not just a ‘how to’ handbook but also an accessible primer in the essentials of kinetic theory & practice."
    —Prof. Michael Geeves, University of Kent

    "This book is a much needed modern update for the underserved field of biomolecular kinetics. It covers the practical ‘how to’ and troubleshooting information for experiments together with the fundamentals in one place. It is written by a genuine expert in kinetic techniques, and is beautifully illustrated. It is highly accessible to readers at all levels, and I highly recommend this text to anyone wishing to learn more about the power and potential applications of kinetics techniques."  
    —Prof. Emma Raven, University of Leicester

    "a gem of a textbook which manages to produce a genuinely fresh, concise yet comprehensive guide through the nuances of biomolecular kinetics, penned by one of the giants of this complex but fundamental field of the life sciences"
    —Prof. Mark Leake, University of York

    "This well written, concise but very comprehensive text will be the go-to book for those wanting to take advantage of new (and classic) approaches, and the challenges and the opportunities they provide."
    —Bob Callender, Professor of Biochemistry, Albert Einstein College of Medicine

    "This is a comprehensive and detailed yet clear and much needed book. Kinetic methods are applied throughout biological sciences and for everyone who is using them this is a must-read."
    —Per Jemth, Department of Medical Biochemistry and Microbiology, Uppsala University

    "Finally, a book that supports a practical entry into kinetics for enzymologists and other biochemists. The field sorely needs a solid, accessible kinetics text like this…."
    —Bruce A. Palfey, Department of Biological Chemistry, University of Michigan Medical School

    "a lively book with a splendid balance between the description of most recent experimental work and easily comprehensible theoretical interpretations…. [It] should help to encourage students to enter this exciting field and help research workers to expand the range of their endeavours."
    —Herbert Gutfreund, University of Bristol